This invention relates to an analytical element for measuring a minute amount of a component in a fluid sample, particularly to an analytical element for analysing a specific minute amount of a component in a biological fluid sample, and an analytical method employing the same.
As a method for detecting a substance contained in an extremely minute amount in a biological fluid sample, various analytical methods have been developed. The analytical methods are based primarily on immune reactions as their principles. While various measurement methods by use of the above principle have been developed, immunoassay is known as the method with the highest precision.
As the immunoassay, radioimmunoassay has been widely employed since Berson and Ylow successed in 1958 in assay of insulin in a serum by use of bovine insulin labelled with radioactive iodine and an anti-insulin antibody in a serum of a diabete patient.
Hereafter, as the labelling compounds, various compounds other than radioisotopes have been developed. As other labelling compounds, there may be included, for example, enzymes, enzyme substrates, coenzymes, enzyme inhibitors, bacteriophages, circulating reactants, metals and organic metallic complexes, organic prosthetic groups, chemiluminescent reactants and fluorescent molecules, etc.
As one of the important problems in technology concerning the above immunoassay, there is a problem how to separate a substance which has been bound (hereinafter abbreviated as B) from a substance which has not been bound (hereinafter abbreviated as F) (hereinafter abbreviated as B/F separation).
In the prior art, various methods have been developed for solving the problems in immunoassay (for example, see Japanese Unexamined patent publication Nos. 38619/1978, 79024/1978, 90859/1980, 67860/1982, 200862/1982, 18167/1983, 77356/1984 and 170768/1984).
However, these methods had the drawbacks that B/F separation is incomplete, that there is a problem with respect to reliability of signal due to much noise, that a substance which can be assayed is limited to low molecular weight substances, etc.
On the other hand, in wet chemistry, there has been developed an immunoassay according to the competitive method by use of a immobilizing phase (see, for example, Japanese Unexamined patent publication Nos. 209994/1983 and 202064/1984). However, in those immunoassays, since the whole enzyme activity is assayed without discrimination between both immobilizing phases, no satisfactory result can be obtained with respect to sensitivity, precision and reproducibility owing to the problems such as backgroun or noise.
On the other hand, in dry chemistry, there has been developed an immunoassay by use of a second antibody (see Japanese Unexamined Patent Publication Nos. 82766/1982 and 82767/1982). However, these techniques involved such problems to be improved that the operation is cumbersome and that a technique for performing spreading with good reproducibility is required, etc. Further, in the invention disclosed in Japanese Unexamined Patent Publication No. 34155/1984, a method by use of an unbound product housing sheet is disclosed. However, even according to this method, the above mentioned problems will occur when measurement is to be performed with the sheet for reaction being contacted with the unbound product housing sheet, and it is also cumbersome to separate the both sheets during measurement, which will particularly become an obstacle when the measurement is automated.
The present invention has been accomplished for the purpose of improving the drawbacks of the prior art as described above, and its object is to provide an analytical element, which is applicable for substances of a broad range of molecular weights (100 to 5,000,000) as the subject to be measured, by performing the B/F separation in a single phase, and can quantitatively determine a specific component in a fluid sample with excellent sensitivity, precision and reproducibility according to a simple operation.